In collaboration with colleagues in Baylor College of Medicine, we explored the feasibility of generating LAA-specific T-cells from stem cell donors of patients with myeloid leukemia. T-cell lines were manufactured from 10 healthy donors by stimulation with 15mer peptide libraries of five LAA: proteinase 3 (Pr3), preferentially expressed antigen in melanoma (PRAME), Wilms tumor gene 1 (WT1), human neutrophil elastase (NE) and melanoma-associated antigen A3,(MAGE) A3. These are known to be expressed in myeloid leukemias and recently shown by us to be well-represented antigens in AML.53 We previously demonstrated that T cells to WT1, PRAME and Proteinase3 (Pr3) increase after HSCT in association with GVL effects.54,55,56 All lines responded to the mix of five LAA and were multi-specific in interferon- ELISpot assay. Although donors showed individual patterns of antigen recognition, all responded comparably to the LAA mix. Known immunogenic peptides of WT1 and Pr3 were identified by epitope mapping in T-cell lines. Experiments showed recognition of partially HLA-matched myeloid leukemia blasts.57 Further studies in ALL showed that WT1-, PRAME- and survivin-specific LAST lysing autologous ALL blasts in vitro could be generated from patients with ALL.58 While most individuals can generate multispecific LAST, the frequencies of these T cells are about one log lower than those observed using viral pepmixes. Furthermore, there is a risk that the pepmix peptides recognized by T cells are not naturally presented by leukemia. To overcome this concern we created full length lentiviral constructs of WT1 and PRAME to transduce DC to stimulate T cells. In comparison with pepmix-generated CTL, lentiviral-DC induced higher frequencies of CD4+ and CD8+ T-cells, which efficiently recognized lentiviral-transduced targets. We have developed conditions for clinical grade cell production of sufficient cell numbers for delivering LAST specific for PRAME, MAGE A4, NyESO and WT1. An NSG mouse model of human acute myeloid leukemia has been established to test the efficacy of adoptively transferred leukemia antigen specific T cells from the original HLA identical donor of the patient's leukemia. In 2017 we completed a series of improvements to the in vitro generation of LAST. This includes selection of naive (CD45+) T cells which contain the LAST precursors and Th17 polarizing conditions which optimize antileukemic cytotoxicity. These LAST are readily expanded to high frequency in culture, kill primary leukemia cells and suppress leukemia in a humanized mouse model. Clinical trials plan to explore safety and efficacy in relapse after HSCT.